Detection of human rhinoviruses by reverse transcription strand invasion based amplification method (RT-SIBA)

Abstract

Background: Rhinovirus (RV), a major cause of respiratory infection in humans, imposes an enormous economic burden due to the direct and indirect costs associated with the illness. Accurate and timely diagnosis is crucial for deciding the appropriate clinical approach and minimizing unnecessary prescription of antibiotics. Diagnosis of RV is extremely challenging due to genetic and serological variability among its numerous types and their similarity to enteroviruses.

Objective: We sought to develop a rapid nucleic acid test that can be used for the detection of Rhinovirus within both laboratory and near patient settings.

Study design: We developed and evaluated a novel isothermal nucleic acid amplification method called Reverse Transcription Strand Invasion-Based Amplification (RT-SIBA) to rapidly detect Rhinovirus from clinical specimens.

Result: The method, RT-SIBA, detected RV in clinical specimens with high analytical sensitivity (96%) and specificity (100%). The time to positive result was significantly shorter for the RV RT-SIBA assay than for a reference RV nucleic acid amplification method (RT-qPCR).

Conclusion: The rapid detection time of the RV SIBA assay, as well as its compatibility with portable instruments, will facilitate prompt diagnosis of infection and thereby improve patient care.

Keywords: Amplification; Diagnostics; Isothermal; Point-of-care; RT-SIBA; Rhinovirus; Virus.

Fig. 1

Rhinovirus amplification by reverse-transcription strand invasion–based amplification (RT-SIBA). 1) Rhinovirus RNA is reverse transcribed to cDNA by the reverse transcriptase enzyme 2) SIBA amplification requires an invasion oligonucleotide (IO) and two target-specific primers. 3) Single strand binding protein, Gp32 binds to oligonucleotides in order to reduce the formation of secondary structures. The recombinase protein, UvsX, coats the IO displacing the bound Gp32. 4) The recombinase-IO complex invades and separates the target duplex. 5) This allows target-specific primers to bind and extend the target via the action of a DNA polymerase. 6) This leads to the synthesis of two copies of the target duplex. 7) The continuous recombinase-mediated target duplex separation and DNA polymerase extension process leads to an exponential amplification under isothermal conditions.

Fig. 2

Optimization of RV SIBA reaction conditions using different invasion oligonucleotide (IO) concentrations. (A) Amplification of 1000 copies of rhinovirus A60 RNA. (B) amplification of 1000 copies of rhinovirus B17 RNA; RV-A-IO, rhinovirus A invasion oligonucleotide; RV-B-IO, rhinovirus B invasion oligonucleotide.

Fig. 3

Sensitivity of RV SIBA assay for detection of rhinoviruses (RVs). (A) rhinovirus A60 RNA. (B) rhinovirus B17 RNA; NTC, no template control.